Abstract Adoptive natural killer (NK) cell immunotherapy using primary NK cells has been challenging, to date. The use of an ”off-the -shelf” NK cell line where multiple genetic changes can be introduced has advantages. In this study, we explored the use of the NK-92MI cell line expressing a chimeric antigen receptor (CAR) targeting human mesothelin, the current target in our human CAR-T cell therapy trials. The NK-92MI cell line (which makes its own IL-2) was purchased from ATCC and transduced with a lentivirus encoding GFP (vector control) or an anti-human mesothelin scFv-41BB-CD3 ζ second-generation CAR (MesoCAR). Addition of IL-21 enhanced transduction. The transduced cells (MesoCar-NK-92MI) were sorted by flow cytometry to achieve an expression level of >98%. The NK markers NKp30, NKp44, NKG2D, 2B4, and CD56 were highly expressed on the Car-NK-92MI cell line (from 75% to 99.9%). To test the antitumor effects in vitro, three types of human tumor lines transduced to express human mesothelin were tested. Lung cancer (A549Meso), mesothelioma (EMMeso), and head and neck cancer (SSC15Meso) cells were exposed to vector control NK-92MI or MesoCar-NK-92MI cells for 22 hours at varying E:T ratios. The supernatants were collected for detection of cytokines and granzyme B. We also determined the killing function of MesoCar-NK-92MI cells after irradiation of the cells at 10 Gy. To test in vivo activity, NSG mice were injected in the flank with 5 million EMMeso cells. After tumors were established, each mouse was intravenously injected with 10 million vector control NK-92MI cells or MesoCar-NK-92MI cells with a boost injection of 5 million effectors on days 4, 8, and 11. Tumor volumes were assessed over time and mice were sacrificed on day 18. In vitro killing results showed augmented antitumor effect in MesoCar-NK-92MI compared to vector control NK-92MI. Irradiated MesoCar-NK-92MI cells still possessed good antitumor activity with only 32% loss of activity on average. Increased cytokine and granzyme B secretion was also seen with the MesoCar-NK-92MI cell line (with or without irradiation) compared to NK-92MI cells. In animal studies, irradiated MesoCar-NK-92MI cells significantly inhibited tumor growth by an average of 59% with no obvious toxicity or tumor growth in other organs. The non-CAR-expressing NK-92MI cells had significantly less antitumor activity. In conclusion, use of an irradiated NK-92MI cell line expressing a CAR targeting human mesothelin could become a safe and possible “off-the-shelf” adoptive cell therapy that could be used alone or in combination with CAR-T cells or other treatments. Citation Format: Jing Sun, Patrick Woodruff, Soyeon Kim, Estela Noguera-Ortega, Evguenia Arguiri, Maria Liousia, Steven Maceko, Edmund Moon, Steven Albelda. Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A67.